The present invention relates to the field of plant molecular biology, more specifically the invention relates to a DNA construct for conferring improved glyphosate tolerance to a wheat plant. The invention more specifically relates to a glyphosate tolerant wheat plant 33391 and progeny thereof and to assays for detecting the presence of wheat plant 33391 DNA in a sample and compositions thereof.
Wheat is an important crop and is a primary food source in many areas of the world. The methods of biotechnology have been applied to wheat for improvement of the agronomic traits and the quality of the product. One such agronomic trait is herbicide tolerance, in particular, tolerance to glyphosate herbicide. This trait in wheat is conferred by the expression of a transgene in the wheat plants (Zhou et al., Plant Cell Rep. 15:159-163, 1995). The expression of foreign genes in plants is known to be influenced by their chromosomal position, perhaps due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulation elements (e.g., enhancers) close to the integration site (Weising et al., Ann. Rev. Genet 22:421-477, 1988). For this reason, it is often necessary to screen a large number of events in order to identify an event characterized by optimal expression of a introduced gene of interest. For example, it has been observed in plants and in other organisms that there may be a wide variation in levels of expression of an introduced gene among events. There may also be differences in spatial or temporal patterns of expression, for example, differences in the relative expression of a transgene in various plant tissues, that may not correspond to the patterns expected from transcriptional regulatory elements present in the introduced gene construct. For this reason, it is common to produce hundreds to thousands of different events and screen those events for a single event that has desired transgene expression levels and patterns for commercial purposes. An event that has desired levels or patterns of transgene expression is useful for introgressing the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny of such crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a number of varieties that are well adapted to local growing conditions.
It would be advantageous to be able to detect the presence of a particular event in order to determine whether progeny of a sexual cross contain a transgene of interest. In addition, a method for detecting a particular event would be helpful for complying with regulations requiring the premarket approval and labeling of foods derived from recombinant crop plants, for example. It is possible to detect the presence of a transgene by any well known nucleic acid detection method such as the polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, etc. As a result, such methods may not be useful for discriminating between different events, particularly those produced using the same DNA construct unless the sequence of chromosomal DNA adjacent to the inserted DNA (xe2x80x9cflanking DNAxe2x80x9d) is known. An event-specific PCR assay is discussed, for example, by Windels et al. (Med. Fac. Landbouww, Univ. Gent 64/5b:459-462, 1999), who identified glyphosate tolerant soybean event 40-3-2 by PCR using a primer set spanning the junction between the insert and flanking DNA, specifically one primer that included sequence from the insert and a second primer that included sequence from flanking DNA.
This invention relates to the improved glyphosate herbicide tolerant wheat (Triticum aestivum) plant 33391 and to a DNA plant expression construct of wheat plant 33391 and the detection of the transgene/genomic insertion region in wheat 33391 and progeny thereof.
According to one aspect of the invention, a DNA construct is provided that when expressed in wheat plant cells and wheat plants confers improved tolerance to glyphosate herbicide. This invention relates to the methods for producing and selecting a glyphosate tolerant wheat plant containing the DNA construct pMON30139. The DNA construct, pMON30139 consists of two transgene expression cassettes. The first expression cassette consists of a rice (Oryzae sativa) actin 1 promoter (P-Os.Act1) and intron (I-Os.Act1) operably joined to an Arabidopsis EPSPS chloroplast transit peptide sequence (TS-At.EPSPS), operably connected to a gene (AGRTU.aroA:CP4) encoding a glyphosate resistant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) isolated from Agrobacterium tumefaciens (AGRTU) sp. strain CP4, operably connected to a nopaline synthase transcriptional terminator (T-AGRTU.nos). The second transgene expression cassette consists of the cauliflower mosaic virus (CaMV) 35S promoter (P-CaMV.35S:en) containing a tandem duplication of the enhancer region, operably connected to a Zea mays Hsp70 intron (I-Zm.Hsp70), operably connected to a nucleic acid sequence encoding an Arabidopsis thaliana EPSPS chloroplast transit peptide sequence, operably connected to a gene encoding a glyphosate resistant 5-enol-pyruvylshikimate-3-phosphate synthase isolated from Agrobacterium tumefaciens sp. strain CP4, operably connected to a nopaline synthase transcriptional terminator. These expression cassettes are in tandem and flanked by DNA regions that contain Agrobacterium tumefaciens DNA sequences (RB and LB) as a components of the process that is used in an Agrobacterium mediated method to insert of the expression cassettes into a wheat genome.
According to another aspect of the invention, wheat 33391 seed comprising such DNA molecules are provided as deposited with the ATCC, accession # PTA-2347. This aspect of the invention thus relates to the seeds of wheat 33391, to the plants of wheat 33391, to the plant parts of wheat 33391 that includes pollen and ovules, and to the methods for producing an improved glyphosate tolerant wheat plant by crossing the wheat plant 33391 with itself or another wheat plant.
According to another aspect of the invention, compositions and methods are provided for detecting the presence of the transgene/genomic insertion region from wheat 33391 plants and seeds. According to one aspect of the invention, DNA molecules are provided that comprise at least one transgene/genomic insertion region sequence of wheat 33391 selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6 and complements thereof, wherein an insertion region sequence spans the junction between heterologous DNA inserted into the wheat genome and DNA from the wheat genome flanking the insertion site and is diagnostic for the event. Included are DNA sequences that comprise a sufficient length of polynucleotides of transgene insert sequence and a sufficient length of polynucleotides of wheat genomic sequence from wheat 33391 of SEQ ID NO:5 that are useful as primer sequences for the production of an amplicon product diagnostic for wheat 33391. Included are DNA sequences that comprise a sufficient length of polynucleotides of transgene insert sequence and a sufficient length of polynucleotides of wheat genomic sequence from wheat 33391 of SEQ ID NO:6 that are useful as primer sequences for the production of an amplicon product diagnostic for wheat 33391.
According to another aspect of the invention DNA molecules are provided that are diagnostic for wheat 33391. This aspect of the invention is directed to the wheat 33391 containing at least one novel DNA molecule. DNA molecules comprising nucleic acid primers are provided that provide at least one novel DNA amplicon product of wheat 33391 consisting of SEQ ID NO:7 and SEQ ID NO:8, or the complements thereof. Such DNA amplicons are diagnostic for wheat 33391. Nucleic acid amplification of genomic DNA of the wheat 33391 produces an amplicon comprising such diagnostic DNA sequences. The invention provides isolated DNA molecules that comprise a sufficient length of transgene insert sequence and a sufficient length of wheat genomic sequence from wheat 33391 to function as primer sequences for the production of an amplicon product diagnostic for wheat 33391.
According to another aspect of the invention, methods of detecting the presence of DNA corresponding to the wheat 33391 in a sample are provided. Such methods comprise: (a) contacting the sample comprising DNA with a primer set that, when used in a nucleic-acid amplification reaction with genomic DNA from wheat 33391, produces an amplicon that is diagnostic for wheat 33391; (b) performing a nucleic acid amplification reaction, thereby producing the amplicon; and (c) detecting the amplicon.
According to another aspect of the invention, a kit is provided for the detection of wheat 33391. The kit includes at least one DNA sequence of sufficient length of polynucleotides complementary to SEQ ID NO:5 or SEQ ID NO:6, wherein the DNA sequences are useful as primers or probes that hybridize to isolated DNA from wheat 33391 or its progeny.
According to another aspect of the invention, methods of producing a wheat plant with improved tolerance to glyphosate are provided that comprise the steps of: (a) sexually crossing a first parental wheat line comprising the pMON30139 construct that confers improved tolerance to application of glyphosate, and a second parental wheat line that lacks glyphosate tolerance, thereby producing a plurality of progeny plants; and (b) selecting a progeny plant that tolerates application of glyphosate. Such methods are useful for introgressing the glyphosate tolerant trait into different genetic backgrounds. Such methods may optionally comprise the further step of back-crossing the progeny plant to the second parental wheat line to produce a wheat plant that tolerates application of glyphosate.